Postnatal cell fates are determined by adult stem cells residing in regenerative tissues such as ectoderm appendages. While their differentiation capacity has been demonstrated extensively, mechanisms that specify cell fate remain poorly understood. Mouse incisor provides an excellent model system to study adult stem cell fate in a regenerating tissue. Incisor stem cells support the continuous growth of the incisors throughout the lifespan of the mouse. Slow dividing dental epithelial stem cells (DE-SC) residing in the cervical loop specify the dental program and regenerate the dental epithelia. The stem cell fate and regeneration are primarily controlled by a specific transcriptional program. We developed a unique stem cell regeneration model, in which the transcriptional program for ectoderm appendages such as tooth and skin is converted. Genomic deletion of one subunit of the transcriptional coactivator Mediator complex, Mediator 1 (Med1), resulted in enamel hypoplasia, in which DE-SCs fail to institute the transcriptional program for a normal dental epithelial fate. Instead, DE-SCs institute an epidermal program and regenerate ectopic hairs in the incisors associated with extended Sox2 expression and reduced Notch signaling. Given these striking preliminary results we hypothesize that the Mediator regulates the transcription factors that control the cell fate of dental stem cells. Med1 deletion alters the pre-existing transcription program for dental fate resulting in a new state of epidermal/hair fate specific transcription. Further study of the Med1 null model will allow us to identify genetic Med1 targets, fate determining transcription factors, through genome wide analyses using microarray and Chip-sequencing (Aim1). We anticipate that these analyses will reveal specific enhancer domains called super-enhancers in which Med1 is highly recruited to organize fate specific chromatin states. We also aim to determine the functionality of these Med1 targets and regulatory molecules including Sox2 and Notch1/Hes1 (Aim2). We will use siRNA mediated gene silencing and overexpression of Med1 and Med1 target genes identified in Aim 1 to determine their role in dental and epidermal cell fate as monitored by lineage specific markers in in vitro culture system. We will then determine their role in 3D co-culture of DE-SC/mesenchyme resembling in vivo stem cell niche system by considering the contribution of dental mesenchyme. Successful completion of this study is expected to uncover a specific transcription program that drives dental cell fate, and more broadly provide insight into the differentiation of ectodermal tissues.